Malaria is the most devastating parasitic disease and, as such, represents one of the most important public health problems worldwide. According to experts in the field, malaria infects 300 million people a year and kills up to 3 million people per year. A vaccine for malaria would drastically reduce the impact of this dangerous disease.
The causative agents in malaria are various species of the eukaryotic genus Plasmodium, including Plasmodium falciparum, Plasmodium vivax, Piasmodium ovale, and Plasmodium Malariae. These parasites have a very complex life cycle that involves both vertebrate and invertebrate hosts. The vertebrate infective form of the parasite (sporozoites) is present in the salivary glands of mosquitoes (typically of the genus Anopheles) and the sporozoites are transferred to humans during feeding by the mosquitoes. In the human host, the sporozoites initially infect the cells of the liver and eventually red blood cells. This infection results in an illness which is potentially fatal to those infected.
Current prophylactic approaches to malaria include the use of drugs, including chloroquine, mefloquine and atovaquone/proquanil. However, multiple drug resistant strains of Plasmodium have recently been observed. In addition, the occurrence of drug resistant strains of malaria is thought to be promoted by the use of these prophylactic antimalarial drugs. Accordingly, significant efforts have been undertaken to develop a vaccine for malaria.
There have been some indications in the scientific literature that a vaccine for malaria could be effective. In regards to a metabolically active non-replicating (attenuated) whole sporozoite vaccine, Nussenzweig and coworkers (Nature 216: 160-162; 1967) reported that immunizing mice with radiation attenuated Plasmodium berghei sporozoites. These rodent studies provided the impetus for human studies, and during the 1970s, Cylde, Rieckmann, and colleagues (Clyde et al.; Am. LT. Med. Sci. 266:169-177; 1973; Clyde et al. Am. LT. Med. Sci. 266:398-401; 1973; Rieckmann et al. Trans. R. Soc. Trop. Med. Hyg. 68:258-259; 1974) conducted limited studies that established that immunizing human volunteers with the bites of irradiated mosquitoes carrying Plasmodium falciparum sporozoites in their salivary glands could protect volunteers against a challenge with fully infectious Plasmodium falciparum sporozoites. Hoffman and Luke (Hoffman et al.; LT. Infect. Dis. 185:1155-1164; 2002) established the full potential of this approach by reporting the results of 10 years' clinical experience with live mosquito immunizations and challenges, and combined their results with all the published clinical reports of immunizing humans with irradiated Plasmodium sporozoites.
The following 3 points summarize the most importaht findings: 1) Thirteen of 14 volunteers immunized by the bites of greater than 1000 infected, irradiated mosquitoes were protected against developing blood-stage P. falciparum infection when challenged within 10 weeks of their last primary immunization; 2) Five of 6 of the 14 volunteers in (1) above when challenged from 23 to 42 weeks (23, 36, 39, 41, and 42 weeks) after their last primary or secondary immunization were protected against experimental challenge; and 3) Seven of seven heterologous challenges (immunized with one strain of P. falciparum and challenged with another strain of P. falciparum) in four individuals were associated with complete protection.
From this, it was demonstrated that protection was achieved in greater than 90% of immunized subjects, lasted for at least 10 months, and demonstrated cross strain (heterologous) protection. For the first time, the true efficacy of this experimental vaccine approach was demonstrated. While this study demonstrated the feasibility of an attenuated malaria vaccine, it was considered for many reasons to be impractical to immunize large numbers of susceptible individuals by employing the bites of irradiated infected mosquitoes.
One technical hurdle to the development of a clinically relevant vaccine is the production of aseptic sporozoites that are free of contamination by unwanted biological agents. Currently, it is not possible to produce Plasmodium falciparum sporozoites using an in vitro process. Therefore, Plasmodium falciparum sporozoites must be obtained from the tissues of infected female Anopheles mosquitoes. However, it is well known that wild and insectary reared mosquitoes are highly contaminated with unwanted biological agents including bacteria, molds, and fungi. This contamination largely prevents the use of mosquito derived parasites in a clinically relevant vaccine suitable fore regulatory licensure. An apparatus and method to produce aseptic Anopheles mosquitoes for the in vivo production of Plasmodium falciparum sporozoites is a critical step in the development of an acceptable attenuated sporozoites vaccine from both a clinical and regulatory perspective.
Contamination of mosquitoes with unwanted biological agents may arise from several sources in the mosquito's life cycle. The surface of mosquito eggs may become contaminated during oviposition from the female mosquito's genital tract and ovipositors. The larvae may retain microbes in their gastrointestinal tract and peritrophic membrane during metamorphosis of larvae to pupae and adult mosquitoes. In addition, multiple environmental factors, including the aquatic habitat of the larvae, the external environment of the adult mosquito, and contaminated skin of an animal upon which the mosquito fed, may contribute to contamination of mosquitoes and thus the Plasmodium parasite.
For decades, non-aseptic sporozoites have been routinely obtained from infected Anopheles mosquitoes for research purposes using labor intensive techniques. There are multiple drawbacks to this standard approach. Since the entire process is conducted under non-sterile conditions, the sporozoites preparation is usually contaminated with microbes. Though sporozoites can be partially purified by a variety of techniques, contamination of the resulting product makes it unsuitable for use in developing a vaccine for human use. Microbially contaminated vaccines can cause an iatrogenic infection of a serious nature in both humans and animals. In addition, the processes of the prior art for rearing non-aseptic mosquitoes are labor intensive and require multiple direct manipulations of the mosquitoes during their life cycle.
Accordingly, one of the limitations in the production of a vaccine for malaria is the ability to obtain a large number of aseptic sporozoites of Plasmodium species. As stated above, sporozoites that are obtained from Anopheles species of mosquitoes using standard techniques results in sporozoites that are not useful in the development of an attenuated sporozoite vaccine. Aseptic sporozoites could be used as a vaccine to generate protective immunological responses safely and efficiently. In addition, the production of such aseptic sporozoites will be a regulatory requirement for the commercial production of a malaria vaccine.
Thus, there has been a long standing need in the medical field for the production of aseptic Plasmodium species. sporozoites and aseptic Anopheles mosquitoes for use in the development of a vaccine for malaria. Apparatuses and methods for the aseptic rearing of Anopheles mosquitoes and Plasmodium parasites can also be used for the aseptic production of other hematophagous insect species and parasites for other critically needed vaccines against parasitic diseases of humans and animals.
In order to develop strains of insects that possess certain desired properties (e.g., hyperinfectivity or hypoallergenicity), it would be useful to employ a device that would allow experiments selectively to evaluate the biting behavior and properties of individual insects. The apparatus would also be useful in the selection of insects that possessed desired properties.
An additional hurdle for the efficient and economical development of an attenuated vaccine for malaria is the deleterious effect that the Plasmodium parasite has on the mosquito host. Anopheles mosquitoes are capable of transmitting Plasmodium sporozoites to a host animal on which they feed. Research indicates that Plasmodium infections of Anopheles female mosquitoes are deleterious to the survival of mosquitoes in both the laboratory and wild-type environment. Thus, the ability to extract large numbers of mosquito phase parasites from female mosquitoes is currently limited by the inability of the mosquitoes to tolerate a heavy Plasmodium parasite burden.
A unique strain of Anopheles mosquito that is tolerant to massive infection with the Plasmodium parasite sporozoites from mosquitoes more efficient. The development of an attenuated Plasmodium sporozoites vaccine derived from this unique strain of Anopheles mosquitoes would thereby be more efficient and economical.
An additional possible difficulty in producing live, attenuated, or killed pathogen vaccines extracted from mosquito tissue is the potential of mosquito antigens to cause hypersensitivity, Arthus, or delayed type hypersensitivity reactions in the inoculated human or animal. Many hypersensitivity salivary antigens in several mosquito species have been identified. These salivary antigens probably confer a survival advantage to wild-type mosquitoes for numerous reasons. However, in laboratory maintained mosquito populations, such antigens are vestiges of their wild ancestors and are probably no longer necessary for selective survival advantage. By developing a technique to create hypoallergenic mosquitoes, it would be possible to create several hypoallergenic mosquito species (Anopheles, Aedes, Culex, etc.) which are specific to a wide variety of mosquito borne infections. These hypoallergenic strains of mosquitoes would have significant utility in the production of safe and effective attenuated parasite vaccines.
Once an attenuated vaccine has been developed, the vaccine should be delivered to an individual in need effectively. However, the standard manner in which vaccines are delivered—bolus injection via a syringe—is not effective in the case of an attenuated malarial vaccine. Vaccines syringes are commonly available in a variety of sizes with an industry standard locking port to which needles of various gauges can be attached. Usually, this system is adequate for typical vaccines, as the required volume is in the range of 500 to 1000 micro liters. Small volume variations from immunization to immunization have no effect on the immunogenicity of the typical vaccine.
However, in some vaccines, such as an attenuated Plasmodium sporozoite vaccine, the inoculum may be to be on the order of a micro liter or less—similar to the injectate of a probing mosquito. Larger volumes may cause the carrying liquid of the vaccine to disturb tissue integrity and cause the liquid to follow tissue planes. In mice, virtually all sporozoite challenge studies and attenuated sporozoites immunization studies are accomplished by intravenous injection of sporozoites, because non-intravenous administration of sporozoites in skin, subcutaneous tissue, and muscle has been associated with much lower infection rates and protection rates. When injected into the skin, sporozoites likely expire within the spaces between tissue planes created by the fluid in which the sporozoites are suspended, providing no opportunity to move throughout the host's cellular structures and into a capillary.
The standard procedure for current vaccines is therefore unsuitable for an ultra-low volume vaccine. To overcome this limitation, one could design a single use micro needle and syringe assembly. While this approach would likely be effective, it would also be impractical as millions of vaccines are to be given in extremely poor countries. Preferably, a malarial vaccine delivery system would be able to both deliver ultra-low volume boluses and also employ standard syringe assemblies as a cost saving measure.
Hurdles also exist for the delivery of frozen attenuated Plasmodium vaccine to individuals survive freezing temperatures in various preservation solutions. The best results, measured after re-warming and estimating the percentage of motile sporozoites and the ability to cause a patent blood stage infection after injection into a study animal, demonstrate that extremely low temperatures (−196 to −70 degrees Celsius) can preserve the sporozoites for many years. However, as preservation temperatures approach zero degrees Celsius, the percentage of viable sporozoites as a function of time drops precipitously in a temperature dependent fashion. This drop in viability limits the utility of malaria vaccines derived from attenuated sporozoites that must retain a high degree of potency during storage and shipment.
The worldwide cold storage and shipment infrastructure is robust and almost all countries have the capability to store relatively large volumes of materials at temperatures approaching zero degrees Celsius. At close to zero degrees Celsius, existing equipment and infrastructure can be adopted to transport an attenuated sporozoite vaccine to even the most remote locations on Earth. A storage and shipment infrastructure that requires temperatures in the range of minus seventy degrees Celsius, though technically feasible, is likely to be routinely possible in only the more technologically advanced nations. However, such extreme cold temperatures require special equipment, care, and will probably entail a large capital expenditure to accommodate the logistics of delivering an attenuated sporozoite vaccine.
The cyropreservation studies of sporozoites have used Plasmodium strains and clones developed for purposes other than developing a freeze tolerant strain of Plasmodium sporozoites. All organisms, to some degree, have the capability to survive temperature variations by utilizing stabilizing proteins, heat shock proteins, sugars, and carbohydrates which prevent the denaturing of critical enzymes, proteins, or limit the damage to cellular substrates caused by ice crystal formation. The malaria parasite, whose life cycle includes passage through the Anopheles mosquito, must have the ability to withstand natural variations in temperature—sometimes quite side as many temperate/tropical climates can vary by thirty to fifty degrees Fahrenheit in a day-night cycle. It can be assumed that genetic variation from Plasmodium organism to organism in nature has led to a wide variation in the ability of sporozoite to withstand temperature extremes.
The key to developing a Plasmodium species better able to survive high temperature cyropreservation conditions is to breed selectively those sporozoites shown to have that capacity. By selecting those sporozoites that survive increasingly high cyropreservation temperatures for longer periods of time while still able to complete a natural life cycle, one can develop a Plasmodium strain that has much greater utility in an attenuated whole-parasite vaccine for worldwide use.